Isolation and antimutagenic activity of some flavanone compounds from Kaempferia rotunda
i n t e r n a t i o n a l j o u r n a l o f c h e m i c a l a n d a n a l y t i c a l s c i e n c e 4 ( 2 0 1 3 ) 3 e8
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/ijcas
Original Article
Isolation and antimutagenic activity of some flavanone
compounds from Kaempferia rotunda
Sri Atun*, Retno Arianingrum, Eddy Sulistyowati, Nurfina Aznam
Department of Chemistry Education, Faculty of Mathematics and Natural Sciences, Yogyakarta State University,
Karangmalang, Depok, Sleman, Yogyakarta 55281, Indonesia
article info
abstract
Article history:
Background/Aims: Kaempferia rotunda (Zingiberaceae), known as kunci pepet or kunir putih
Received 9 February 2013
in Indonesia, has been traditionally used in abdominal pain, sputum laxative, wounds and
Accepted 18 March 2013
diarrhea colic disorder. This study was conducted to isolate and to investigate anti-
Available online 24 March 2013
mutagenic activity of some flavanones from K. rotunda.
Methods: The milled dried rhizoma of K. rotunda (3 kg) was extracted exhaustively with
Keywords:
methanol. The methanol extract was partitionated three times by n-hexane, chloroform,
Antimutagenic
and ethyl acetate respectively. Each fraction was fractionated by vacuum liquid chroma-
Kaempferia rotunda
tography (VLC) and purified by column chromatography gravitation. Identification struc-
Flavanone
tures of all pure compounds were elucidated based on spectroscopic methods (UV, IR, and
NMR) and compared to the spectroscopic previously reported data. Antimutagenic activity
test was observed in vivo based on the number of micronucleated polychromatic cell
erythrocytes (MNPCE) from male Balb-c mice (8e12 week) induced by cyclophosphamide.
Results: From the dried and milled rhizoma of K. rotunda, three known flavanones, namely
5-hydroxy-7-methoxyflavanone (1), 7-hydroxy-5-methoxyflavanone (2), and 5,7-dihydroxyflavanone (3) were isolated. The methanol extract and isolated flavanones from K. rotunda
showed significant antimutagenic effect compared to control group.
Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights
reserved.
1.
Introduction
Cancer becomes a significant health problem in the world.
According to WHO in 2000e2010 cancer ranks as the second
cause of death worldwide after heart disease and in the 2030 is
expected to increase and become the first ranks.1 Cancer is the
uncontrolled growth of cells, followed by cell invasion into the
surrounding tissue and spread to other body parts. The main
characteristic of cancer is a continuous cell proliferation,
causing an imbalance between life and death cells.2 Cancer is
a multi-factorial, multi-stage and multimechanistic complex
process with multiple risk factors that involve interplay between genetic and environmental components.3 One of the
factors that cause cancer is the mutation in a DNA gene. When
a mutation is happen in the DNA, then the cancer may be very
difficult to cure. The mutations in somatic cells are not only
involved in the carcinogenesis process but also play a role in
the pathogenesis of other chronic degenerative diseases, such
* Corresponding author. Fax: þ62 (0) 274540713.
E-mail addresses: Atun_1210@yahoo.com, sriatun@uny.ac.id (S. Atun).
0976-1209/$ e see front matter Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.ijcas.2013.03.004
4
i n t e r n a t i o n a l j o u r n a l o f c h e m i c a l a n d a n a l y t i c a l s c i e n c e 4 ( 2 0 1 3 ) 3 e8
as atherosclerosis and heart diseases, which are the leading
causes of death in the human population.4 The mechanism of
the mutation could be spontaneously and by the induction of
some factors such as radiation, chemicals, and viruses.
Mutagen is a substance that causes mutations, whereas
compounds that can inhibit the mutation called
antimutagenic.5
There is considerable evidence that the effects of mutagenic and carcinogenic agents can be altered by many dietary
constituents or natural bioactive materials in many plant
species. Investigations of antimutagenic potentials of herbal
used in traditional medicine are generating great interest with
the growing evidence of their safe consumption. Some herbs
that have been studied as antimutagenic among others
Momordica charantia,5 ascorbic acid,6 several compounds curcumin and its derivatives,7 phenolic compounds such as
ellagic acid,8 and polyphenols in fruits, vegetables, and tea.9
Kaempferia genus is perennial member of the Zingiberaceae
family and is cultivated in Indonesia and other parts of
Southeast Asia. Number of studies has been conducted,
providing information related to Kaempferia as chemopreventive agent. The methanol extract of Kaempferia parviflora showed a high cytotoxic activity against human
cholangiocarcinoma (HuCCA-1 and RMCCA-1).10 Some compounds such as panduratin A from Kaempferia pandurata
showed high cytotoxic activity against human epidermis KB
cancer cells,11 showed high toxicity against human pancreatic
cancer cells Panc-1.12 The methanol extract of K. parviflora are
also induced apoptosis of the cancer cells HL-60.13 Previous
studies showed that some compounds that have cytotoxic
activity against cancer cells also showed antimutagenic
properties.4 This paper will report our investigation of some
flavanones from Kaempferia rotunda and their antimutagenic
activity.
2.
Material and method
2.1.
Apparatus
UV and IR spectra were measured with Varian Cary 100 Conc
and Shimadzu 8300 FTIR, respectively. 1H and 13C NMR spectra
were recorded with Jeol JNM A-5000 spectrometers, operating
at 500.0 MHz (1H) and 125.0 MHz (13C) using residual and
deuterated solvent peaks as internal standards. Evaporator
Buchi Rotavapor R-114, vacuum liquid chromatography (VLC)
was carried out using Si-gel Merck 60 GF254 (230e400 mesh),
column chromatography using Si-gel Merck 60 (200e400
mesh), and TLC analysis on precoated Si gel plates Merck
Kieselgel 60 F254 0.25 mm, 20 20 cm, water bath, shaker
bath, microscup, camera, counter, deskglasser, eppendorf,
object glass, and analytical balance.
2.2.
2.3.
Plant materials
Samples of the rizhoma of K. rotunda were collected in
December 2010 from the Merapi Farma, Yogyakarta,
Indonesia. The plant was identified by the staff at the Faculty
Biology, Gadjah Mada University, Indonesia and a voucher
specimen (KR-01e2012) was deposited at the Organic Laboratory, Yogyakarta State University, Indonesia.
2.4.
Animal test
The experiment were carried out on adult male Balb-c mice
(8e12 week) obtained from LPPT, Gadjah Mada University,
Indonesia. All mice, 2e3 month old, weighed between 22, 5
and 27 g and were kept under constant environmental conditions with a 12: 12 lightedark cycle, at 23e25 C room temperature. The animals were fed standard granulated chow
(pelet 789) and had access to drinking water ad libitum. Animal
experiments were done in accordance with Institutional Protocols of animal care. The mice were divided into ten groups
consisting of six animal each. Group one served as normal
control, group two was treated with cyclophosphamide doses
at 50 mg/kg BW each day. Group three and four were treated
with sample methanol extract K. rotunda doses at 300 and
600 mg/kg BW each day per oral, whereas group five until ten
each group were treated with flavanone compounds (A, B, C)
dose at 30 and 60 mg/kg BW daily per oral. Group three until
ten at 30 min after treated with compounds followed cyclophosphamide doses at 50 mg/kg BW daily by intravenal. After
30 h treatment bone marrow from all the mice was collected
respectively.
2.5.
Antimutagenic assay
The antimutagenic assay of this experiment was determined
by bone marrow micronucleus assay.14 After 30 h the mice
were anesthetized and the bone marrow was aspirated from
femur and tibia into one ml of 1% physiological salin. The cell
suspension was centrifuge (1000 rpm for 5 min) and the
smears were prepared from the pellet on chemically cleaned
glass slides and washed with ethanol absolute at 10 min,
dried, and stained with Giemsa stain. To detect possible
micronucleus, the portion of micronucleated polychromatic
erythrocytes (MNPCE) in 1000 erythrocytes/mice was calculated by using a light microscope (1000 magnification).
The frequency of MNPCE in individual mice was used as
the experimental unit, with standard deviation based on
difference among mice within the same group. The data
from the micronucleus assay were statistically analyzed
using Student’s t-test, comparing the treated groups with
control and significance level considered was p < 0.05. The
percentage of antimutagenic activity calculated by the
following formula:
Chemicals
Sodium carboxyl methyl cellulose (Na-CMC), cyclophosphamide monohydrate, physiological salin, xilol, Giemsa stain,
were obtained from E. Merck in pure analytical grade. Several
solvent such as chloroform, hexane, ethyl acetate, acetone,
ethanol, and methanol.
%Activity ¼
ðmean CP ðmean S þ mean NÞÞ
100%
ðmean CP mean NÞ
Where CP ¼ positive control group treated with cyclophosphamide; N ¼ negative control group; S ¼ group treated with
methanol extracts or flavanones (A, B, or C)
5
i n t e r n a t i o n a l j o u r n a l o f c h e m i c a l a n d a n a l y t i c a l s c i e n c e 4 ( 2 0 1 3 ) 3 e8
2.6.
Isolation and identification structure
The milled dried rhizoma of K. rotunda (3 kg) was extracted
exhaustively with methanol. The methanol extract on removal
of the solvent under reduced pressure gave a brown residue
(230 g). A portion (200 g) of the total methanol extract was
partitionated three times by n-hexane, chloroform, and ethyl
acetate respectively. Each fractions was evaporated to dryness
under vacuum to yield brown residue n-hexane fraction (40 g),
chloroform fraction (120 g), and ethyl acetate fractions (31 g). A
portion (25 g) ethyl acetate fraction was fractionated by vacuum liquid chromatography (VLC) (silica gel GF 60 Merck 250 g;
f: 10 cm, t ¼ 10 cm), using n-hexane, n-hexane-ethyl acetate
(9:1; 8:2; 6:4; 5:5; 4:6; and 6:4), ethyl acetate, acetone, and
methanol of increasing polarity as eluents to give twenty
fractions. These fractions were combined based on the same
TLC profiles and evaporated to give three major fractions A
(1e5) (3.5 g), B (6e15) (10 g), and C (16e20) (6.6 g). Fraction A was
recrystallized using methanol to give colorless crystal of 5hydroxy-7-methoxyflavanone (1) (1.5 g). Fraction B was purified by column chromatography gravitation using Si-gel Merck
60 (200e400 mesh), (f: 1.5 cm, t ¼ 15 cm) eluted with hexaneethyl acetate (6:4) as solvent to give 48 fraction. Fraction
(13e21) were combined and evaporated to give a pale yellow
crystal of 7-hydroxy-5-methoxyflavanone (2) (1.2 g). Fraction
(30e45) yielded as yellow needle shaped crystals of 5,7dihydroxyflavanone (3) (1.8 g).
Residue of n-hexane fraction (40 g), was recrystallized
using methanol to give colorless crystal of 5-hydroxy-7methoxyflavanone (1) (4, 5 g). Chloroform fraction (60 g) was
fractionated by VLC (silica gel GF 60 Merck 250 g; f: 10 cm,
t ¼ 10 cm), using n-hexane, n-hexane-ethyl acetate (9:1; 8:2;
6:4; 5:5; 4:6; and 6:4), ethyl acetate, acetone, and methanol of
increasing polarity as eluents to give twenty fractions. Fraction (4e10) yielded colorless crystal of 5-hydroxy-7methoxyflavanone (1) (3.0 g), whereas fraction (15e18) yielded
as
yellow
needle
shaped
crystals
of
5,7dihydroxyflavanone (3) (2.5 g). The structures of these compounds (1e3) were established on the basis of their spectral
data, including UV, IR and NMR one and two dimension HMQC
and HMBC. A complete spectral data are included in the supplementary information (Fig. 3e14).
5-Hydroxy-7-methoxyflavanone (1) was obtained as a
colorless crystal. The UV (in methanol solvent) lmax: 213;
287 nm. The IR (KBr pellet) ymax: 3444; 1645; 1621; 1581; 1381;
and 1158 cm-1. 1H NMR (500 MHz, CDCl3) : d 3.08 (1H, dd, 2.8;
12.0); 2, 84 (1H, d, 2.8); 3.81 (3H, s); 5.43 (1H, d, 12.0); 6.04 (1H, br
s); 6.06 (1H, br s); 7.42 (2H, br s); 7.43 (3H, br s); and 12.03 (1H, s,
OH) ppm. 13C NMR (125 MHz, CDCl3): d 43.5; 55.85; 77.45; 94.43;
95.3; 103.3; 126.3 (3C); 129.0 (2C); 138.54; 163.14; 164.3; 168.3;
195.93 ppm. The result of this analysis compound 1 using NMR
one and two dimension spectrophotometer listed in Table 1.
7-Hydroxy-5-methoxyflavanone (2) was obtained as a pale
yellow crystal. The UV (in methanol solvent) lmax : 227;
288 nm. The IR (KBr pellet) ymax: 3450; 1645; 1621; 1581; 1381;
and 1158 cm-1. 1H NMR (500 MHz, Acetone-d6): d 2.67 (1H,d, br
s); 2.97 (1H, d, 12.6); 3.79 (3H, s); 5.48 (1H, d, 12.6); 6.09 (1H, br s);
6, 15 (1H, d, 2.5); 7.54 (2H, d, 8.6); 7.43 (2H, t, 8.6; 8.0); 7.37 (1H, t,
8.0; 8.0); 9.42 (1H, br s, OH). 13C NMR (125 MHz, Acetone-d6):
d 46.48; 56.14; 79.80; 94.23; 96.65; 106.18; 127.23 (2C); 129.22;
Table 1 e 1H NMR, 13C NMR, and HMBC data of compound
1 (CDCl3).
P
No
d C ppm
D H ( H; m; J Hz)
HMBC (H/C)
1
2
3
4
5
6
7
8
9
10
10
20
30
40
50
60
5-OH
7-OCH3
e
77.45
43.50
195.93
164.30
95.30
168.30
94.43
163.14
103.30
138.54
126.30
129.00
126.30
129.00
126.30
e
55.85
5.43 (1H, d, 12.0)
3.08 (1H, dd, 2.8; 12.0);
2.84 (1H, d, 2.8)
e
e
6.04 (1H, br s)
e
6.06 (1H, br s)
e
e
e
7.43 (1H, br s)
7.42 (1H, br s)
7.43 (1H, br s)
7.42 (1H, br s)
7.43 (1H, br s)
12.03 (1H, s)
3.81 (3H, s)
C4; C10 ; C3
C4; C2
C5; C7
C8; C5
C10; C5
C10 ; C2
C6; C10; C5
129.47 (2C); 140.67; 163.79; 165.06; 165.60; 187.84 ppm. The
result of this analysis compound 2 using NMR one and two
dimension spectrophotometer listed in Table 2.
5,7-Dihydroxyflavanone (3) was obtained as a yellow needle shaped crystals. UV (in methanol solvent) lmax : 210;
288 nm. The IR (KBr pellet) ymax: 3444; 1631; 1583; 1488; 1302;
1168; 1089 cm-1. 1H NMR (500 MHz, Acetone-d6) : d 2.82 (1H, dd,
2.9; 12.6); 3.18 (1H, dd, 2.9; 12.6); 5.56 (1H, dd, 2.9; 12.6); 5.98 (1H,
d, 2.5); 6.01 (1H, br s); 7.42 (1H, t, 8.0; 8.0); 7.45 (2H, t, 8.0; 8.0);
7.56 (2H, d, 8.0); 9.63 (1H, br s, OH); 12.16 (1H, br s, OH) ppm. 13C
NMR (125 MHz, Acetone-d6): d 43.63; 79.47; 95.91; 96.96; 103.29;
127.32 (2C); 129.45 (2C); 129.51; 140.06; 164.19; 165.33; 167.38;
196.85 ppm. The result of this analysis compound 3 using NMR
one and two dimension spectrophotometer listed in Table 3.
Table 2 e 1H NMR, 13C NMR, and HMBC data of compound
2 (Acetone-d6).
P
No
d C ppm
D H ( H; m; J Hz)
HMBC
1
2
3
4
5
6
7
8
9
10
10
20
30
40
50
60
7-OH
5-OCH3
79.80
46.48
187.84
163.73
96.65
165.60
94.23
165.06
106.18
140.67
127.23
129.47
129.22
129.47
127.43
e
56.14
5.48 (1H, d, 12.6)
2.67 (1H, br s);
2.97 (1H, d, 12.6)
e
C10 ; C3; C4
C4; C2
6, 15 (1H, d, 2.5)
e
6.09 (1H, br s)
e
e
e
7.54 (1H, d, 8.6)
7.43 (1H, t, 8.6; 8.0)
7.37 (1H,t, 8.0; 8.0)
7.43 (1H, t, 8.6; 8.0)
7.54 (1H, d, 8.6)
9.42 (br s)
3.79 (s)
C8; C5
e
C10; C9
e
C10 ; C2
C10 ; C20
C20
C10 ; C20
C10 ; C2
C8: C6
6
i n t e r n a t i o n a l j o u r n a l o f c h e m i c a l a n d a n a l y t i c a l s c i e n c e 4 ( 2 0 1 3 ) 3 e8
Table 3 e 1H NMR, 13C NMR, and HMBC data of compound
3 (Acetone-d6).
P
No
d C ppm
D H ( H; m; J Hz)
HMBC
1
2
3
e
79.47
43.63
4
5
6
7
8
9
10
10
20
30
40
50
60
5-OH
7-OH
196.85
165.33
96.60
164.19
95.91
167.38
103.29
140.06
127.32
129.45
129.51
129.45
127.32
3.
5.56 (1H, dd, 2.9; 12.6)
2.82 (1H, dd, 2.9; 12.6);
3.18 (1H, dd, 2.9; 12.6)
e
e
5.98 (1H, d, 8.0)
e
6.01 (1H, br s)
e
e
e
7.56 (1H, d, 8.0)
7.45 (1H, t, 8.0; 8.0)
7.42 (1H, t, 8.0; 8.0)
7.45 (1H, t, 8.0; 8.0)
7.56 (1H, d, 8.0)
12.16 (1H, br s)
9.63 (1H, br s)
C4; C1’; C3
C4; C2
C4
C10; C5
C10 ; C20
C20 ; C1
C30 ; C20
C20 ; C10
C10 ; C20
C10; C6
Result and discussion
Characterization of isolated three flavanones by using NMR
are listed in Tables 1e3. Structure of the isolated compounds
at Fig. 1.
5-Hydroxy-7-methoxyflavanone (1) was obtained as a
colorless crystal. Its UV (in methanol solvent) spectrum
showed absorption maximum at 213; 287 nm suggesting the
phenol ring. The IR (KBr pellet) spectrum exhibited hydroxyl
group (3440 cme1), C]O carbonyl (1645 cm-1), C]C aromatic
(1621; 1581 cme1), and CeOeC bond (1158 cme1), these spectral
characteristic absorptions supporting (1) to be a phenolic
compound. 13C NMR spectra showed one signals for carbon
carbonyl at d 195.93, three signal oxyaryl carbon at d 168.3 (C7), 164.3 (C-5), and 163.14 (C-9) ppm, nine aromatic carbon at
d 95.30 (C-6), 94.43 (C-8), 103.3 (C-10), 138.54 (C-10 ), 126.30 (C-20 ,
C-40 & C60 ), 129.00 (C-3 & C-5) ppm. Additionally, the 13C NMR
also exhibited one oxyalkyl carbon at d 77.45 (C-2) ppm,
aliphatic carbon at d 43.50 (C-3) ppm, and one methoxyl carbon at d 55.85 (OCH3). The 1H NMR spectrum of (1) in CDCl3
exhibited signals for a monosubstituted phenyl ring d 7.43 (3H,
br s, H-20 , H-60 , and H-40 ) & 7.42 (2H, br s, H30 & H50 ) ppm. The 1H
3'
2'
4'
1
O
8
R1
7
6
1'
5'
9
2
10 4
3
6'
5
R2
O
(1) R1 = OCH3; R2 = OH
(2) R1 = OH ; R2 = OCH3
(3) R1 = OH ; R2 = OH
Fig. 1 e Structure of the isolated flavanones from K. rotunda.
NMR spectrum also showed one of meta-coupled aromatic
protons signals at d 6.04 (1H, br s, H-6) and 6.06 (1 H, br s, H-8)
ppm, oxyalkyl proton at d 5.43 (1H, d, 12.0 Hz, H-2), and
aliphatic proton at d 3.08 (1H, dd, 2.8; 12.0 Hz, H-3a) and d 2.84
(1H, d, 2.8 Hz, H-3b). Additionally, the 1H NMR spectrum
exhibited signals for methoxyl group at d 3.81 (3H, s), and
hydroxyl proton at d 12.03 (1 H, s). The connection between
protons and their corresponding carbons was established by
HMQC. Further support for the structure (1) was obtained from
HMBC measurement (Table 1). These results suggested that
compound (1) was a flavanone with substituted methoxyl and
hydroxyl group. Further evidence for the structure assigned to
compound (1) came from comparison of their spectral data
with those reported in the literature. Therefore, it may be
conclude that compound (1) is pinostrobin that isolated from
K. pandurata.15
7-Hydroxy-5-methoxyflavanone (2) was obtained as a pale
yellow crystal. The UV (in methanol solvent) lmax : 227;
288 nm. The IR (KBr pellet) spectrum exhibited hydroxyl group
(3450 cme1), C]O carbonyl (1645 cm-1), C]C aromatic (1621;
1581 cme1), and CeOeC bond (1158 cme1). The 1H and 13C NMR
spectrum showed the data that is very similar to compound 1.
The 1H NMR spectrum of (2) in Acetone-d6 exhibited proton
signals for a monosubstituted phenyl ring at d 7.54 (2H, d,
J ¼ 8.6 Hz, H-20 , H-60 ), 7.43 (2H, t, J ¼ 8.6; 8.0 Hz, H30 & H50 ), and
7.37 (1H, t, J ¼ 8.0) ppm. The 1H NMR spectrum also showed
one of meta-coupled aromatic protons signals at d 6.15 (1H, br
s, H-6) and 6.09 (1H, br s, H-8) ppm, oxyalkyl proton at d 5.48
(1H, d, 12.6 Hz, H-2) ppm, aliphatic proton at d 2.97 (1H, d,
12.6 Hz, H-3a) and d 2.67 (1H, d, 2.8 Hz, H-3b) ppm, methoxyl
group at d 3.79 (3H, s) ppm, and hydroxyl proton at d 9.42 (br s)
ppm. 13C NMR spectra showed sixteen carbon consisting of
carbonyl carbon at d 187.84, three signal oxyaryl carbon at
d 163.73 (C-5), 165.60 (C-7), and 165.06 (C-9) ppm, nine aromatic
carbon at d 96.65 (C-6), 94.23 (C-8), 106.18 (C-10), 140.67 (C-10 ),
127.23 (C-20 , C60 ), 129.47 (C-3, C-5), 129.47 (C-40 ), one oxyalkyl
carbon at d 79.80 (C-2), alkyl carbon at d 46.48 (C-3), and one
methoxyl carbon at d 56.14 (OCH3) ppm. The HMQC spectrum
supported complete assignment of all proton-bearing carbon
signals of compound 2 (Table 2). Further support for the
structure 2 was obtained from HMBC measurement. Compound 2 is flavanone with methoxyl group at C-5 and hydroxyl
group at C-7. It is based from the HMBC spectrum showed a
correlation between proton hydroxyl with C8 and C6, and
there is no correlation between hydroxyl with C10, such as
those in compound 1. Therefore, compound 2 is 7-Hydroxy-5methoxyflavanone. These compound have the similar NMR
data with alpinetin that isolated from K. pandurata.15
5,7-Dihydroxyflavanone (3) was obtained as a yellow needle shaped crystals. UV (in methanol solvent)l max : 210;
288 nm. The IR (KBr pellet) spectrum exhibited hydroxyl group
(3444 cm-1). Carbonyl group (1631 cm-1), C]C aromatic (1583;
1488 cm-1), and CeOeC group (1302 cm-1). 1H NMR (500 MHz,
Acetone-d6) showed aliphatic proton at d 2.82 (1H, dd, J ¼ 2.9;
12.6 Hz, H-3a); 3.18 (1H, dd, J ¼ 2.9; 12.6 Hz, H-3b), oxyalkyl
proton at d 5.56 (1H, dd, J ¼ 2.9; 12.6 Hz), one benzene ring with
meta-coupled aromatic protons signals at d 5.98 (1H, d,
J ¼ 2.5 Hz) and 6.01 (1H, br s); and five proton signals for a
monosubstituted phenyl ring at d 7.42 (1H, t, J ¼ 8.0; 8.0 Hz, H40 ), 7.45 (2H, t, J ¼ 8.0; 8.0 Hz, H 30 & 50 ); and 7.56 (2H, d, J ¼ 8.0 Hz,
7
i n t e r n a t i o n a l j o u r n a l o f c h e m i c a l a n d a n a l y t i c a l s c i e n c e 4 ( 2 0 1 3 ) 3 e8
Table 4 e Percentage antimutagenic activity of these groups.
No
1
2
3
4
5
6
7
8
9
10
Groups treatment
Mean of
MNPCE SD
% Antimutagenic
activity
Negative control (blanco) (Na-CMC 1%)
Positive control (cyclophosphamide dose 50 mg/kg BW)
Methanol extract dose 300 mg/kg bw followed cyclophosphamide 50 mg/kg BW
Methanol extract dose 600 mg/kg BW followed cyclophosphamide 50 mg/kg BW
5-hydroxy-7-methoxyflavanone (A1) dose 30 mg/kg BW followed cyclophosphamide
50 mg/kg BW
5- hydroxy-7-methoxyflavanone (A2) dose 60 mg/kg BW followed cyclophosphamide
50 mg/kg BW
7-hydroxy-5-methoxyflavanone (B1) dose 30 mg/kg BW, followed cyclophosphamide
50 mg/kg BW
7-hydroxy-5-methoxyflavanone (B2) dose 60 mg/kg BW, followed cyclophosphamide
50 mg/kg BW
5,7-dihydroxyflavanone (C1) dose 30 mg/kg BW followed cyclophosphamide 50 mg/
kg BW
5,7-dihydroxyflavanone (C2) dose 60 mg/kg BW followed cyclophosphamide 50 mg/
kg BW
0.0 0.0
5.75 3.4
2.25 1.7
1.0 1.4
2.5 3.0
e
e
55.0
80.0
56.5
H-20 & H-60 ); and two proton signal from hydroxyl group at
d 9.63 (1H, br s, OH); 12.16 (1H, br s, OH) ppm. 13C NMR
(125 MHz, Acetone-d6) showed sixteen carbon consisting of
aliphatic carbon at d 43.63; oxyalkyl carbon at d 79.47 ppm,
aromatic carbon at d 95.91; 96.96; 103.29; 127.32 (2C); 129.45
(2C); 129.51; and 140.06 ppm, oxyaryl carbon at d 164.19; 165.33;
and 167.38 ppm, and carbon carbonyl at d 196.85 ppm. The
connection between protons and their corresponding carbons
was established by HMQC. Further support for the structure (3)
was obtained from HMBC measurement (Table 3). These results suggested that compound (3) was a flavanone with
substituted two hydroxyl group. Therefore, compound 3 is 5,7dihydroxyflavanone. These compound have the similar NMR
data with pinocembrin that isolated from K. pandurata.15
The antimutagenic assay of this experiment was determined by bone marrow micronucleus assay.14 The bone
marrow micronucleus test is one of the most suitable antimutagenic assay by in vivo. The antimutagenic activity of
methanol extract and isolated flavanone compounds was
evaluated by measuring their inhibitory effect on cyclophosphamide induced mutagenesis. The oral administration of 30
and 60 mg/kg BW of flavanone prior to cyclophosphamide
exposure reduced the frequency of MNPCE in all groups studied.
The results as shown in Table 4, demonstrated that the flavanone isolated from K. rotunda at a dose of 30 mg/kg BW, which is
0.2 0.44
96.5
0.2 0.44
96.5
0.0 0.0
100
0.4 0.89
93.0
0.0 0.0
100
5-hydroxy-7-methoxyflavanone (A1), 5,7-dihydroxyflavanone
(C1), 7-hydroxy-5-methoxyflavanone (B1), have antimutagenic
activity percentage of 56.5%; 93.0% and 96.5% respectively.
Meanwhile, at a dose of 60 mg/kg BW, three compounds showed
a very high activity. Methanol extract of K. rotunda which is a
crude extract contain a mixture of some flavanone compounds.
It showed significant activity but lower than pure compounds.
The polychromatic erythrocytes can be showed at Fig. 2.
The protection mechanism against mutagenic agent has not
been known yet, however, it may scavenge free radicals or
inhibiting DNA strand breaks or may enhance DNA repair
mechanism. Micronucleus is one indicator of DNA mutation.
Micronucleus is the result of a broken chromosome, and then
appears as a small nucleus in the cell. The micronucleus derived
from acentric fragments or lagging chromosome which only
appear in the anaphase stage of the mitosis process. So that the
micronucleus are easily observed in the cells that constantly
divide, such as cells in the bone marrow. The number of
micronucleated polychromatic erythrocytes (MNPCE) shows
the level of genetic damage in the erythropoiesis process.
Alkylation on agent such as cyclophosphamide has
cytotoxic effects through the transfer of alkyl cluster to
various elements of the cell. Alkylation of DNA in the nucleus is a major mechanism leads to the cell death.
Cyclophosphamide is converted by cytochrome P450
Fig. 2 e The normal polychromatic erythrocytes (A) and MNPCE (B).
8
i n t e r n a t i o n a l j o u r n a l o f c h e m i c a l a n d a n a l y t i c a l s c i e n c e 4 ( 2 0 1 3 ) 3 e8
isoenzymes into 4-hydroxycyclophosphamide in the liver.
4-hydroxycyclophosphamide is oxidized into aldophosphamide as the active metabolites. Furthermore, aldophosphamide will break down into phosphamide mustard and
acrolein, which is highly toxic. This mechanism of toxicity
happens in the bone marrow.
Based on the antimutagenic activity and molecular structure relationship, known that the hydroxyl group at position
C-7 can increase antimutagenic activity. This is related to the
nature of the two compounds flavanones polarity which is
having hydroxyl group at position C-7. Compound 5-hydroxy7-methoxyflavanone or pinostrobin do not have a hydroxyl
group at position C-7, but has a hydroxyl group at position C-5.
Hydroxyl group at position C-5 is not in the free state, because
it can form hydrogen bonds with the carbonyl group (C3).
Edenharder R, et al16 reported that the correlation of antimutagenicity of natural compounds of plant origin was
equivalent with polarity of flavonols.
Although the biochemical mechanisms underlying flavanone compounds activities are not yet clear, our results
demonstrated that K. rotunda has a preventive effect against
chromosome fragmentation in vivo, probably due to its free
radical scavenging capability. Our observations are consistent
with those in the other studies. Moreover, study showed that
phenolic compounds have bioactivity as antioxidant, antimutagenic, and chemopreventive. Therefore, our results
confirm and extend our knowledge on the ability of methanol
extract and isolated flavanone compounds from K. rotunda to
protect DNA, showing that both prevent chromosome damage
after cyclophosphamide exposure in mice.
4.
Conclusion
From the dried and milled rhizoma of K. rotunda, three known
flavanones, namely 5-hydroxy-7-methoxyflavanone (1), 7hydroxy-5-methoxyflavanone (2), and 5,7-dihydroxyflavanone
(3) were isolated. The methanol extract and isolated flavanones
from K. rotunda showed significant antimutagenic effect
compared to control group.
Conflicts of interest
All authors have none to declare.
Acknowledgments
The financial support from research grand of Yogyakarta State
University, Indonesia is acknowledged.
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Antimutagenic effects of natural phenolic compounds in
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10. Leardkamolkarn V, Sunida T, Bung-orn S. Pharmacological
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12. Nwet Nwet W, Suresh A, Hiroyasu E, Yasuhiro T,
Shigetoshi K. Panduratins DdI, novel secondary metabolites
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15. Ching AYL, Wah TS, Sukari MA, Lian GEC, Rahmani M,
Khalid K. Characterization of flavanoid derivative from
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16. Edenharder R, Tang X. Inhibition of mutagenicity of 2nitrofluorene, 3-nitrofluorene and 1-nitropyrene by
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Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/ijcas
Original Article
Isolation and antimutagenic activity of some flavanone
compounds from Kaempferia rotunda
Sri Atun*, Retno Arianingrum, Eddy Sulistyowati, Nurfina Aznam
Department of Chemistry Education, Faculty of Mathematics and Natural Sciences, Yogyakarta State University,
Karangmalang, Depok, Sleman, Yogyakarta 55281, Indonesia
article info
abstract
Article history:
Background/Aims: Kaempferia rotunda (Zingiberaceae), known as kunci pepet or kunir putih
Received 9 February 2013
in Indonesia, has been traditionally used in abdominal pain, sputum laxative, wounds and
Accepted 18 March 2013
diarrhea colic disorder. This study was conducted to isolate and to investigate anti-
Available online 24 March 2013
mutagenic activity of some flavanones from K. rotunda.
Methods: The milled dried rhizoma of K. rotunda (3 kg) was extracted exhaustively with
Keywords:
methanol. The methanol extract was partitionated three times by n-hexane, chloroform,
Antimutagenic
and ethyl acetate respectively. Each fraction was fractionated by vacuum liquid chroma-
Kaempferia rotunda
tography (VLC) and purified by column chromatography gravitation. Identification struc-
Flavanone
tures of all pure compounds were elucidated based on spectroscopic methods (UV, IR, and
NMR) and compared to the spectroscopic previously reported data. Antimutagenic activity
test was observed in vivo based on the number of micronucleated polychromatic cell
erythrocytes (MNPCE) from male Balb-c mice (8e12 week) induced by cyclophosphamide.
Results: From the dried and milled rhizoma of K. rotunda, three known flavanones, namely
5-hydroxy-7-methoxyflavanone (1), 7-hydroxy-5-methoxyflavanone (2), and 5,7-dihydroxyflavanone (3) were isolated. The methanol extract and isolated flavanones from K. rotunda
showed significant antimutagenic effect compared to control group.
Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights
reserved.
1.
Introduction
Cancer becomes a significant health problem in the world.
According to WHO in 2000e2010 cancer ranks as the second
cause of death worldwide after heart disease and in the 2030 is
expected to increase and become the first ranks.1 Cancer is the
uncontrolled growth of cells, followed by cell invasion into the
surrounding tissue and spread to other body parts. The main
characteristic of cancer is a continuous cell proliferation,
causing an imbalance between life and death cells.2 Cancer is
a multi-factorial, multi-stage and multimechanistic complex
process with multiple risk factors that involve interplay between genetic and environmental components.3 One of the
factors that cause cancer is the mutation in a DNA gene. When
a mutation is happen in the DNA, then the cancer may be very
difficult to cure. The mutations in somatic cells are not only
involved in the carcinogenesis process but also play a role in
the pathogenesis of other chronic degenerative diseases, such
* Corresponding author. Fax: þ62 (0) 274540713.
E-mail addresses: Atun_1210@yahoo.com, sriatun@uny.ac.id (S. Atun).
0976-1209/$ e see front matter Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.ijcas.2013.03.004
4
i n t e r n a t i o n a l j o u r n a l o f c h e m i c a l a n d a n a l y t i c a l s c i e n c e 4 ( 2 0 1 3 ) 3 e8
as atherosclerosis and heart diseases, which are the leading
causes of death in the human population.4 The mechanism of
the mutation could be spontaneously and by the induction of
some factors such as radiation, chemicals, and viruses.
Mutagen is a substance that causes mutations, whereas
compounds that can inhibit the mutation called
antimutagenic.5
There is considerable evidence that the effects of mutagenic and carcinogenic agents can be altered by many dietary
constituents or natural bioactive materials in many plant
species. Investigations of antimutagenic potentials of herbal
used in traditional medicine are generating great interest with
the growing evidence of their safe consumption. Some herbs
that have been studied as antimutagenic among others
Momordica charantia,5 ascorbic acid,6 several compounds curcumin and its derivatives,7 phenolic compounds such as
ellagic acid,8 and polyphenols in fruits, vegetables, and tea.9
Kaempferia genus is perennial member of the Zingiberaceae
family and is cultivated in Indonesia and other parts of
Southeast Asia. Number of studies has been conducted,
providing information related to Kaempferia as chemopreventive agent. The methanol extract of Kaempferia parviflora showed a high cytotoxic activity against human
cholangiocarcinoma (HuCCA-1 and RMCCA-1).10 Some compounds such as panduratin A from Kaempferia pandurata
showed high cytotoxic activity against human epidermis KB
cancer cells,11 showed high toxicity against human pancreatic
cancer cells Panc-1.12 The methanol extract of K. parviflora are
also induced apoptosis of the cancer cells HL-60.13 Previous
studies showed that some compounds that have cytotoxic
activity against cancer cells also showed antimutagenic
properties.4 This paper will report our investigation of some
flavanones from Kaempferia rotunda and their antimutagenic
activity.
2.
Material and method
2.1.
Apparatus
UV and IR spectra were measured with Varian Cary 100 Conc
and Shimadzu 8300 FTIR, respectively. 1H and 13C NMR spectra
were recorded with Jeol JNM A-5000 spectrometers, operating
at 500.0 MHz (1H) and 125.0 MHz (13C) using residual and
deuterated solvent peaks as internal standards. Evaporator
Buchi Rotavapor R-114, vacuum liquid chromatography (VLC)
was carried out using Si-gel Merck 60 GF254 (230e400 mesh),
column chromatography using Si-gel Merck 60 (200e400
mesh), and TLC analysis on precoated Si gel plates Merck
Kieselgel 60 F254 0.25 mm, 20 20 cm, water bath, shaker
bath, microscup, camera, counter, deskglasser, eppendorf,
object glass, and analytical balance.
2.2.
2.3.
Plant materials
Samples of the rizhoma of K. rotunda were collected in
December 2010 from the Merapi Farma, Yogyakarta,
Indonesia. The plant was identified by the staff at the Faculty
Biology, Gadjah Mada University, Indonesia and a voucher
specimen (KR-01e2012) was deposited at the Organic Laboratory, Yogyakarta State University, Indonesia.
2.4.
Animal test
The experiment were carried out on adult male Balb-c mice
(8e12 week) obtained from LPPT, Gadjah Mada University,
Indonesia. All mice, 2e3 month old, weighed between 22, 5
and 27 g and were kept under constant environmental conditions with a 12: 12 lightedark cycle, at 23e25 C room temperature. The animals were fed standard granulated chow
(pelet 789) and had access to drinking water ad libitum. Animal
experiments were done in accordance with Institutional Protocols of animal care. The mice were divided into ten groups
consisting of six animal each. Group one served as normal
control, group two was treated with cyclophosphamide doses
at 50 mg/kg BW each day. Group three and four were treated
with sample methanol extract K. rotunda doses at 300 and
600 mg/kg BW each day per oral, whereas group five until ten
each group were treated with flavanone compounds (A, B, C)
dose at 30 and 60 mg/kg BW daily per oral. Group three until
ten at 30 min after treated with compounds followed cyclophosphamide doses at 50 mg/kg BW daily by intravenal. After
30 h treatment bone marrow from all the mice was collected
respectively.
2.5.
Antimutagenic assay
The antimutagenic assay of this experiment was determined
by bone marrow micronucleus assay.14 After 30 h the mice
were anesthetized and the bone marrow was aspirated from
femur and tibia into one ml of 1% physiological salin. The cell
suspension was centrifuge (1000 rpm for 5 min) and the
smears were prepared from the pellet on chemically cleaned
glass slides and washed with ethanol absolute at 10 min,
dried, and stained with Giemsa stain. To detect possible
micronucleus, the portion of micronucleated polychromatic
erythrocytes (MNPCE) in 1000 erythrocytes/mice was calculated by using a light microscope (1000 magnification).
The frequency of MNPCE in individual mice was used as
the experimental unit, with standard deviation based on
difference among mice within the same group. The data
from the micronucleus assay were statistically analyzed
using Student’s t-test, comparing the treated groups with
control and significance level considered was p < 0.05. The
percentage of antimutagenic activity calculated by the
following formula:
Chemicals
Sodium carboxyl methyl cellulose (Na-CMC), cyclophosphamide monohydrate, physiological salin, xilol, Giemsa stain,
were obtained from E. Merck in pure analytical grade. Several
solvent such as chloroform, hexane, ethyl acetate, acetone,
ethanol, and methanol.
%Activity ¼
ðmean CP ðmean S þ mean NÞÞ
100%
ðmean CP mean NÞ
Where CP ¼ positive control group treated with cyclophosphamide; N ¼ negative control group; S ¼ group treated with
methanol extracts or flavanones (A, B, or C)
5
i n t e r n a t i o n a l j o u r n a l o f c h e m i c a l a n d a n a l y t i c a l s c i e n c e 4 ( 2 0 1 3 ) 3 e8
2.6.
Isolation and identification structure
The milled dried rhizoma of K. rotunda (3 kg) was extracted
exhaustively with methanol. The methanol extract on removal
of the solvent under reduced pressure gave a brown residue
(230 g). A portion (200 g) of the total methanol extract was
partitionated three times by n-hexane, chloroform, and ethyl
acetate respectively. Each fractions was evaporated to dryness
under vacuum to yield brown residue n-hexane fraction (40 g),
chloroform fraction (120 g), and ethyl acetate fractions (31 g). A
portion (25 g) ethyl acetate fraction was fractionated by vacuum liquid chromatography (VLC) (silica gel GF 60 Merck 250 g;
f: 10 cm, t ¼ 10 cm), using n-hexane, n-hexane-ethyl acetate
(9:1; 8:2; 6:4; 5:5; 4:6; and 6:4), ethyl acetate, acetone, and
methanol of increasing polarity as eluents to give twenty
fractions. These fractions were combined based on the same
TLC profiles and evaporated to give three major fractions A
(1e5) (3.5 g), B (6e15) (10 g), and C (16e20) (6.6 g). Fraction A was
recrystallized using methanol to give colorless crystal of 5hydroxy-7-methoxyflavanone (1) (1.5 g). Fraction B was purified by column chromatography gravitation using Si-gel Merck
60 (200e400 mesh), (f: 1.5 cm, t ¼ 15 cm) eluted with hexaneethyl acetate (6:4) as solvent to give 48 fraction. Fraction
(13e21) were combined and evaporated to give a pale yellow
crystal of 7-hydroxy-5-methoxyflavanone (2) (1.2 g). Fraction
(30e45) yielded as yellow needle shaped crystals of 5,7dihydroxyflavanone (3) (1.8 g).
Residue of n-hexane fraction (40 g), was recrystallized
using methanol to give colorless crystal of 5-hydroxy-7methoxyflavanone (1) (4, 5 g). Chloroform fraction (60 g) was
fractionated by VLC (silica gel GF 60 Merck 250 g; f: 10 cm,
t ¼ 10 cm), using n-hexane, n-hexane-ethyl acetate (9:1; 8:2;
6:4; 5:5; 4:6; and 6:4), ethyl acetate, acetone, and methanol of
increasing polarity as eluents to give twenty fractions. Fraction (4e10) yielded colorless crystal of 5-hydroxy-7methoxyflavanone (1) (3.0 g), whereas fraction (15e18) yielded
as
yellow
needle
shaped
crystals
of
5,7dihydroxyflavanone (3) (2.5 g). The structures of these compounds (1e3) were established on the basis of their spectral
data, including UV, IR and NMR one and two dimension HMQC
and HMBC. A complete spectral data are included in the supplementary information (Fig. 3e14).
5-Hydroxy-7-methoxyflavanone (1) was obtained as a
colorless crystal. The UV (in methanol solvent) lmax: 213;
287 nm. The IR (KBr pellet) ymax: 3444; 1645; 1621; 1581; 1381;
and 1158 cm-1. 1H NMR (500 MHz, CDCl3) : d 3.08 (1H, dd, 2.8;
12.0); 2, 84 (1H, d, 2.8); 3.81 (3H, s); 5.43 (1H, d, 12.0); 6.04 (1H, br
s); 6.06 (1H, br s); 7.42 (2H, br s); 7.43 (3H, br s); and 12.03 (1H, s,
OH) ppm. 13C NMR (125 MHz, CDCl3): d 43.5; 55.85; 77.45; 94.43;
95.3; 103.3; 126.3 (3C); 129.0 (2C); 138.54; 163.14; 164.3; 168.3;
195.93 ppm. The result of this analysis compound 1 using NMR
one and two dimension spectrophotometer listed in Table 1.
7-Hydroxy-5-methoxyflavanone (2) was obtained as a pale
yellow crystal. The UV (in methanol solvent) lmax : 227;
288 nm. The IR (KBr pellet) ymax: 3450; 1645; 1621; 1581; 1381;
and 1158 cm-1. 1H NMR (500 MHz, Acetone-d6): d 2.67 (1H,d, br
s); 2.97 (1H, d, 12.6); 3.79 (3H, s); 5.48 (1H, d, 12.6); 6.09 (1H, br s);
6, 15 (1H, d, 2.5); 7.54 (2H, d, 8.6); 7.43 (2H, t, 8.6; 8.0); 7.37 (1H, t,
8.0; 8.0); 9.42 (1H, br s, OH). 13C NMR (125 MHz, Acetone-d6):
d 46.48; 56.14; 79.80; 94.23; 96.65; 106.18; 127.23 (2C); 129.22;
Table 1 e 1H NMR, 13C NMR, and HMBC data of compound
1 (CDCl3).
P
No
d C ppm
D H ( H; m; J Hz)
HMBC (H/C)
1
2
3
4
5
6
7
8
9
10
10
20
30
40
50
60
5-OH
7-OCH3
e
77.45
43.50
195.93
164.30
95.30
168.30
94.43
163.14
103.30
138.54
126.30
129.00
126.30
129.00
126.30
e
55.85
5.43 (1H, d, 12.0)
3.08 (1H, dd, 2.8; 12.0);
2.84 (1H, d, 2.8)
e
e
6.04 (1H, br s)
e
6.06 (1H, br s)
e
e
e
7.43 (1H, br s)
7.42 (1H, br s)
7.43 (1H, br s)
7.42 (1H, br s)
7.43 (1H, br s)
12.03 (1H, s)
3.81 (3H, s)
C4; C10 ; C3
C4; C2
C5; C7
C8; C5
C10; C5
C10 ; C2
C6; C10; C5
129.47 (2C); 140.67; 163.79; 165.06; 165.60; 187.84 ppm. The
result of this analysis compound 2 using NMR one and two
dimension spectrophotometer listed in Table 2.
5,7-Dihydroxyflavanone (3) was obtained as a yellow needle shaped crystals. UV (in methanol solvent) lmax : 210;
288 nm. The IR (KBr pellet) ymax: 3444; 1631; 1583; 1488; 1302;
1168; 1089 cm-1. 1H NMR (500 MHz, Acetone-d6) : d 2.82 (1H, dd,
2.9; 12.6); 3.18 (1H, dd, 2.9; 12.6); 5.56 (1H, dd, 2.9; 12.6); 5.98 (1H,
d, 2.5); 6.01 (1H, br s); 7.42 (1H, t, 8.0; 8.0); 7.45 (2H, t, 8.0; 8.0);
7.56 (2H, d, 8.0); 9.63 (1H, br s, OH); 12.16 (1H, br s, OH) ppm. 13C
NMR (125 MHz, Acetone-d6): d 43.63; 79.47; 95.91; 96.96; 103.29;
127.32 (2C); 129.45 (2C); 129.51; 140.06; 164.19; 165.33; 167.38;
196.85 ppm. The result of this analysis compound 3 using NMR
one and two dimension spectrophotometer listed in Table 3.
Table 2 e 1H NMR, 13C NMR, and HMBC data of compound
2 (Acetone-d6).
P
No
d C ppm
D H ( H; m; J Hz)
HMBC
1
2
3
4
5
6
7
8
9
10
10
20
30
40
50
60
7-OH
5-OCH3
79.80
46.48
187.84
163.73
96.65
165.60
94.23
165.06
106.18
140.67
127.23
129.47
129.22
129.47
127.43
e
56.14
5.48 (1H, d, 12.6)
2.67 (1H, br s);
2.97 (1H, d, 12.6)
e
C10 ; C3; C4
C4; C2
6, 15 (1H, d, 2.5)
e
6.09 (1H, br s)
e
e
e
7.54 (1H, d, 8.6)
7.43 (1H, t, 8.6; 8.0)
7.37 (1H,t, 8.0; 8.0)
7.43 (1H, t, 8.6; 8.0)
7.54 (1H, d, 8.6)
9.42 (br s)
3.79 (s)
C8; C5
e
C10; C9
e
C10 ; C2
C10 ; C20
C20
C10 ; C20
C10 ; C2
C8: C6
6
i n t e r n a t i o n a l j o u r n a l o f c h e m i c a l a n d a n a l y t i c a l s c i e n c e 4 ( 2 0 1 3 ) 3 e8
Table 3 e 1H NMR, 13C NMR, and HMBC data of compound
3 (Acetone-d6).
P
No
d C ppm
D H ( H; m; J Hz)
HMBC
1
2
3
e
79.47
43.63
4
5
6
7
8
9
10
10
20
30
40
50
60
5-OH
7-OH
196.85
165.33
96.60
164.19
95.91
167.38
103.29
140.06
127.32
129.45
129.51
129.45
127.32
3.
5.56 (1H, dd, 2.9; 12.6)
2.82 (1H, dd, 2.9; 12.6);
3.18 (1H, dd, 2.9; 12.6)
e
e
5.98 (1H, d, 8.0)
e
6.01 (1H, br s)
e
e
e
7.56 (1H, d, 8.0)
7.45 (1H, t, 8.0; 8.0)
7.42 (1H, t, 8.0; 8.0)
7.45 (1H, t, 8.0; 8.0)
7.56 (1H, d, 8.0)
12.16 (1H, br s)
9.63 (1H, br s)
C4; C1’; C3
C4; C2
C4
C10; C5
C10 ; C20
C20 ; C1
C30 ; C20
C20 ; C10
C10 ; C20
C10; C6
Result and discussion
Characterization of isolated three flavanones by using NMR
are listed in Tables 1e3. Structure of the isolated compounds
at Fig. 1.
5-Hydroxy-7-methoxyflavanone (1) was obtained as a
colorless crystal. Its UV (in methanol solvent) spectrum
showed absorption maximum at 213; 287 nm suggesting the
phenol ring. The IR (KBr pellet) spectrum exhibited hydroxyl
group (3440 cme1), C]O carbonyl (1645 cm-1), C]C aromatic
(1621; 1581 cme1), and CeOeC bond (1158 cme1), these spectral
characteristic absorptions supporting (1) to be a phenolic
compound. 13C NMR spectra showed one signals for carbon
carbonyl at d 195.93, three signal oxyaryl carbon at d 168.3 (C7), 164.3 (C-5), and 163.14 (C-9) ppm, nine aromatic carbon at
d 95.30 (C-6), 94.43 (C-8), 103.3 (C-10), 138.54 (C-10 ), 126.30 (C-20 ,
C-40 & C60 ), 129.00 (C-3 & C-5) ppm. Additionally, the 13C NMR
also exhibited one oxyalkyl carbon at d 77.45 (C-2) ppm,
aliphatic carbon at d 43.50 (C-3) ppm, and one methoxyl carbon at d 55.85 (OCH3). The 1H NMR spectrum of (1) in CDCl3
exhibited signals for a monosubstituted phenyl ring d 7.43 (3H,
br s, H-20 , H-60 , and H-40 ) & 7.42 (2H, br s, H30 & H50 ) ppm. The 1H
3'
2'
4'
1
O
8
R1
7
6
1'
5'
9
2
10 4
3
6'
5
R2
O
(1) R1 = OCH3; R2 = OH
(2) R1 = OH ; R2 = OCH3
(3) R1 = OH ; R2 = OH
Fig. 1 e Structure of the isolated flavanones from K. rotunda.
NMR spectrum also showed one of meta-coupled aromatic
protons signals at d 6.04 (1H, br s, H-6) and 6.06 (1 H, br s, H-8)
ppm, oxyalkyl proton at d 5.43 (1H, d, 12.0 Hz, H-2), and
aliphatic proton at d 3.08 (1H, dd, 2.8; 12.0 Hz, H-3a) and d 2.84
(1H, d, 2.8 Hz, H-3b). Additionally, the 1H NMR spectrum
exhibited signals for methoxyl group at d 3.81 (3H, s), and
hydroxyl proton at d 12.03 (1 H, s). The connection between
protons and their corresponding carbons was established by
HMQC. Further support for the structure (1) was obtained from
HMBC measurement (Table 1). These results suggested that
compound (1) was a flavanone with substituted methoxyl and
hydroxyl group. Further evidence for the structure assigned to
compound (1) came from comparison of their spectral data
with those reported in the literature. Therefore, it may be
conclude that compound (1) is pinostrobin that isolated from
K. pandurata.15
7-Hydroxy-5-methoxyflavanone (2) was obtained as a pale
yellow crystal. The UV (in methanol solvent) lmax : 227;
288 nm. The IR (KBr pellet) spectrum exhibited hydroxyl group
(3450 cme1), C]O carbonyl (1645 cm-1), C]C aromatic (1621;
1581 cme1), and CeOeC bond (1158 cme1). The 1H and 13C NMR
spectrum showed the data that is very similar to compound 1.
The 1H NMR spectrum of (2) in Acetone-d6 exhibited proton
signals for a monosubstituted phenyl ring at d 7.54 (2H, d,
J ¼ 8.6 Hz, H-20 , H-60 ), 7.43 (2H, t, J ¼ 8.6; 8.0 Hz, H30 & H50 ), and
7.37 (1H, t, J ¼ 8.0) ppm. The 1H NMR spectrum also showed
one of meta-coupled aromatic protons signals at d 6.15 (1H, br
s, H-6) and 6.09 (1H, br s, H-8) ppm, oxyalkyl proton at d 5.48
(1H, d, 12.6 Hz, H-2) ppm, aliphatic proton at d 2.97 (1H, d,
12.6 Hz, H-3a) and d 2.67 (1H, d, 2.8 Hz, H-3b) ppm, methoxyl
group at d 3.79 (3H, s) ppm, and hydroxyl proton at d 9.42 (br s)
ppm. 13C NMR spectra showed sixteen carbon consisting of
carbonyl carbon at d 187.84, three signal oxyaryl carbon at
d 163.73 (C-5), 165.60 (C-7), and 165.06 (C-9) ppm, nine aromatic
carbon at d 96.65 (C-6), 94.23 (C-8), 106.18 (C-10), 140.67 (C-10 ),
127.23 (C-20 , C60 ), 129.47 (C-3, C-5), 129.47 (C-40 ), one oxyalkyl
carbon at d 79.80 (C-2), alkyl carbon at d 46.48 (C-3), and one
methoxyl carbon at d 56.14 (OCH3) ppm. The HMQC spectrum
supported complete assignment of all proton-bearing carbon
signals of compound 2 (Table 2). Further support for the
structure 2 was obtained from HMBC measurement. Compound 2 is flavanone with methoxyl group at C-5 and hydroxyl
group at C-7. It is based from the HMBC spectrum showed a
correlation between proton hydroxyl with C8 and C6, and
there is no correlation between hydroxyl with C10, such as
those in compound 1. Therefore, compound 2 is 7-Hydroxy-5methoxyflavanone. These compound have the similar NMR
data with alpinetin that isolated from K. pandurata.15
5,7-Dihydroxyflavanone (3) was obtained as a yellow needle shaped crystals. UV (in methanol solvent)l max : 210;
288 nm. The IR (KBr pellet) spectrum exhibited hydroxyl group
(3444 cm-1). Carbonyl group (1631 cm-1), C]C aromatic (1583;
1488 cm-1), and CeOeC group (1302 cm-1). 1H NMR (500 MHz,
Acetone-d6) showed aliphatic proton at d 2.82 (1H, dd, J ¼ 2.9;
12.6 Hz, H-3a); 3.18 (1H, dd, J ¼ 2.9; 12.6 Hz, H-3b), oxyalkyl
proton at d 5.56 (1H, dd, J ¼ 2.9; 12.6 Hz), one benzene ring with
meta-coupled aromatic protons signals at d 5.98 (1H, d,
J ¼ 2.5 Hz) and 6.01 (1H, br s); and five proton signals for a
monosubstituted phenyl ring at d 7.42 (1H, t, J ¼ 8.0; 8.0 Hz, H40 ), 7.45 (2H, t, J ¼ 8.0; 8.0 Hz, H 30 & 50 ); and 7.56 (2H, d, J ¼ 8.0 Hz,
7
i n t e r n a t i o n a l j o u r n a l o f c h e m i c a l a n d a n a l y t i c a l s c i e n c e 4 ( 2 0 1 3 ) 3 e8
Table 4 e Percentage antimutagenic activity of these groups.
No
1
2
3
4
5
6
7
8
9
10
Groups treatment
Mean of
MNPCE SD
% Antimutagenic
activity
Negative control (blanco) (Na-CMC 1%)
Positive control (cyclophosphamide dose 50 mg/kg BW)
Methanol extract dose 300 mg/kg bw followed cyclophosphamide 50 mg/kg BW
Methanol extract dose 600 mg/kg BW followed cyclophosphamide 50 mg/kg BW
5-hydroxy-7-methoxyflavanone (A1) dose 30 mg/kg BW followed cyclophosphamide
50 mg/kg BW
5- hydroxy-7-methoxyflavanone (A2) dose 60 mg/kg BW followed cyclophosphamide
50 mg/kg BW
7-hydroxy-5-methoxyflavanone (B1) dose 30 mg/kg BW, followed cyclophosphamide
50 mg/kg BW
7-hydroxy-5-methoxyflavanone (B2) dose 60 mg/kg BW, followed cyclophosphamide
50 mg/kg BW
5,7-dihydroxyflavanone (C1) dose 30 mg/kg BW followed cyclophosphamide 50 mg/
kg BW
5,7-dihydroxyflavanone (C2) dose 60 mg/kg BW followed cyclophosphamide 50 mg/
kg BW
0.0 0.0
5.75 3.4
2.25 1.7
1.0 1.4
2.5 3.0
e
e
55.0
80.0
56.5
H-20 & H-60 ); and two proton signal from hydroxyl group at
d 9.63 (1H, br s, OH); 12.16 (1H, br s, OH) ppm. 13C NMR
(125 MHz, Acetone-d6) showed sixteen carbon consisting of
aliphatic carbon at d 43.63; oxyalkyl carbon at d 79.47 ppm,
aromatic carbon at d 95.91; 96.96; 103.29; 127.32 (2C); 129.45
(2C); 129.51; and 140.06 ppm, oxyaryl carbon at d 164.19; 165.33;
and 167.38 ppm, and carbon carbonyl at d 196.85 ppm. The
connection between protons and their corresponding carbons
was established by HMQC. Further support for the structure (3)
was obtained from HMBC measurement (Table 3). These results suggested that compound (3) was a flavanone with
substituted two hydroxyl group. Therefore, compound 3 is 5,7dihydroxyflavanone. These compound have the similar NMR
data with pinocembrin that isolated from K. pandurata.15
The antimutagenic assay of this experiment was determined by bone marrow micronucleus assay.14 The bone
marrow micronucleus test is one of the most suitable antimutagenic assay by in vivo. The antimutagenic activity of
methanol extract and isolated flavanone compounds was
evaluated by measuring their inhibitory effect on cyclophosphamide induced mutagenesis. The oral administration of 30
and 60 mg/kg BW of flavanone prior to cyclophosphamide
exposure reduced the frequency of MNPCE in all groups studied.
The results as shown in Table 4, demonstrated that the flavanone isolated from K. rotunda at a dose of 30 mg/kg BW, which is
0.2 0.44
96.5
0.2 0.44
96.5
0.0 0.0
100
0.4 0.89
93.0
0.0 0.0
100
5-hydroxy-7-methoxyflavanone (A1), 5,7-dihydroxyflavanone
(C1), 7-hydroxy-5-methoxyflavanone (B1), have antimutagenic
activity percentage of 56.5%; 93.0% and 96.5% respectively.
Meanwhile, at a dose of 60 mg/kg BW, three compounds showed
a very high activity. Methanol extract of K. rotunda which is a
crude extract contain a mixture of some flavanone compounds.
It showed significant activity but lower than pure compounds.
The polychromatic erythrocytes can be showed at Fig. 2.
The protection mechanism against mutagenic agent has not
been known yet, however, it may scavenge free radicals or
inhibiting DNA strand breaks or may enhance DNA repair
mechanism. Micronucleus is one indicator of DNA mutation.
Micronucleus is the result of a broken chromosome, and then
appears as a small nucleus in the cell. The micronucleus derived
from acentric fragments or lagging chromosome which only
appear in the anaphase stage of the mitosis process. So that the
micronucleus are easily observed in the cells that constantly
divide, such as cells in the bone marrow. The number of
micronucleated polychromatic erythrocytes (MNPCE) shows
the level of genetic damage in the erythropoiesis process.
Alkylation on agent such as cyclophosphamide has
cytotoxic effects through the transfer of alkyl cluster to
various elements of the cell. Alkylation of DNA in the nucleus is a major mechanism leads to the cell death.
Cyclophosphamide is converted by cytochrome P450
Fig. 2 e The normal polychromatic erythrocytes (A) and MNPCE (B).
8
i n t e r n a t i o n a l j o u r n a l o f c h e m i c a l a n d a n a l y t i c a l s c i e n c e 4 ( 2 0 1 3 ) 3 e8
isoenzymes into 4-hydroxycyclophosphamide in the liver.
4-hydroxycyclophosphamide is oxidized into aldophosphamide as the active metabolites. Furthermore, aldophosphamide will break down into phosphamide mustard and
acrolein, which is highly toxic. This mechanism of toxicity
happens in the bone marrow.
Based on the antimutagenic activity and molecular structure relationship, known that the hydroxyl group at position
C-7 can increase antimutagenic activity. This is related to the
nature of the two compounds flavanones polarity which is
having hydroxyl group at position C-7. Compound 5-hydroxy7-methoxyflavanone or pinostrobin do not have a hydroxyl
group at position C-7, but has a hydroxyl group at position C-5.
Hydroxyl group at position C-5 is not in the free state, because
it can form hydrogen bonds with the carbonyl group (C3).
Edenharder R, et al16 reported that the correlation of antimutagenicity of natural compounds of plant origin was
equivalent with polarity of flavonols.
Although the biochemical mechanisms underlying flavanone compounds activities are not yet clear, our results
demonstrated that K. rotunda has a preventive effect against
chromosome fragmentation in vivo, probably due to its free
radical scavenging capability. Our observations are consistent
with those in the other studies. Moreover, study showed that
phenolic compounds have bioactivity as antioxidant, antimutagenic, and chemopreventive. Therefore, our results
confirm and extend our knowledge on the ability of methanol
extract and isolated flavanone compounds from K. rotunda to
protect DNA, showing that both prevent chromosome damage
after cyclophosphamide exposure in mice.
4.
Conclusion
From the dried and milled rhizoma of K. rotunda, three known
flavanones, namely 5-hydroxy-7-methoxyflavanone (1), 7hydroxy-5-methoxyflavanone (2), and 5,7-dihydroxyflavanone
(3) were isolated. The methanol extract and isolated flavanones
from K. rotunda showed significant antimutagenic effect
compared to control group.
Conflicts of interest
All authors have none to declare.
Acknowledgments
The financial support from research grand of Yogyakarta State
University, Indonesia is acknowledged.
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